Understanding key regulators of normal skeletal function have been facilitated by the study of osteogenesis imperfecta (01) or brittle bone disease. Our genetic studies have shown that humans with loss of function mutations in FKBPIO (which encodes the FKBP65 protein) have progressive deforming 01, which may also be associated with progressive joint contractures. What remains unresolved, but necessary to understand, are the cellular consequences and molecular interactions by which FKBP65 regulates synthesis of type I procollagen and formation of a proper bone extracellular matrix (ECM) leading to normal mineralization. The Specific Aims are: 1. Determine the consequences of FKBP10 loss on cellular phenotype. Loss of FKBP65 has significant effects on cellular phenotype indicating increased ER stress. We will determine the molecular basis for these observations in human 01 cells. 2. Determine the in vivo role of FKBP65 in mesenchymally derived tissues and determine its interaction with other ER-localized proteins. FkbpIO conditional knockout mice have been generated to determine whether the abnormal human cellular phenotype is recapitulated in the mouse. Based on the human phenotype, we will determine tissue-specific roles for Fkbp65 in the FkbpIO null mice.. 3. Determine the molecular basis for other recessive forms of 01. Using autozygosity mapping we have identified three new loci for recessively inherited forms of 01. We hypothesize that, like most genes associated with 01, these genes will be involved in the processing of type I procollagen and that their identification will reveal additional components essential for bone formation. We will use exome sequencing of genes in the autozygous regions to define these new 01 associated genes. The work proposed will determine the role of FKBP10/FKBP65 in mesenchymal tissues and will define new components necessary for normal bone formation. Overall the data generated will determine the molecules necessary for the synthesis, mineralization and maintenance of a normal type I collagen extracellular matrix. RELEVANCE (See instructions): The results of this project will have an immediate impact by providing improved genetic counseling and diagnostic testing for families with brittle bone disease. Determining the mechanism of disease in these new forms of Ol develop will identify disease-based strategies and specific molecular targets for therapy.